Several histochemical staining protocols, including Hematoxylin and Eosin (H&E) staining and Papanicolaou (PAP) staining, rely on the dye hematoxylin to stain cytological and tissue samples. In particular, hematoxylin staining of cell nuclei is used by pathologists to detect the presence of malignant and/or metastatic cells in a tumor biopsy sample.
Hematoxylin is a naturally-occurring compound found in the red heartwood of trees of the genus Hematoxylon. Hematoxylin itself is colorless in aqueous solution and is not the active ingredient that stains tissue components. Rather, an oxidation product of hematoxylin, hematein, becomes the active staining component of a hematoxylin dye solution, particularly upon complexation with a mordant. Hematein is produced naturally through exposure to air and sunlight. The natural process is termed “ripening,” and can take 3 or more months to provide a solution suitable for staining cells.
In order to accelerate the conversion of hematoxylin to hematein, a chemical oxidant can be utilized. Unfortunately, the accelerated process often produces ineffective reaction products such as oxyhematein and complex polymeric precipitates, and also provides a solution that degrades faster than a naturally ripened dye solution. The exact amount of oxidant needed to quantitatively oxidize hematoxylin to hematein can be used to help avoid over-oxidation to ineffective products, but a partially-oxidized solution is more typically utilized when staining is not performed immediately. In a partially-oxidized solution, natural oxidation of the hematoxylin that is remaining after a chemical oxidation step will continue to replace any hematein that is either consumed during staining or is naturally oxidized further to ineffective products. Still, the concentration (and amount) of hematein can change over time.
Since hematein is the active staining component of a hematoxylin solution, changes in its concentration (and/or the concentration of its mordant complexes) over time leads to staining inconsistencies. In a manual staining procedure, changes in hematein content of a hematoxylin solution can be compensated for by altering the contact time of a biological sample with the solution based on visual inspection. For example, an apparently under-stained sample can simply be placed back into the hematoxylin solution for a period of time to increase the staining intensity. In an automated staining procedure, however, “visual” inspection and extension of the exposure time in response to under-staining can require costly imaging equipment and can disrupt processing of other samples. Therefore, a need exists for a hematoxylin solution wherein the concentration of hematein available for staining is better stabilized over time.